The present invention relates to a method for producing a nucleic acid immobilized carrier in which many nucleic acid probes having prescribed nucleotide sequences different from each other are immobilized on a substrate.
Genetic screening technologies using a DNA array have recently attracted remarkable attention (Beattie et al. 1993, Fodor et al. 1991, Khrapko et al. 1989, Southern et al. 1994). This DNA array comprises a carrier which carries 101 to 105 types of DNA probes differing in sequence, said probes being immobilized on the surface of a glass substrate or silicon substrate which is several centimeters by several centimeters square. The DNA array greatly contributes to recent development of genetic analysis technologies. The outline of its principle is summarized as follows.
First, on the DNA array, the DNA probe is reacted with a sample gene which is labeled with a fluorescent dye, radioactive isotope (RI) or the like, thereby to be coupled by hybridization with the sample gene which has a sequence complementary to that of the DNA probe. This ensures that when the sample gene has a sequence complementary to that of the DNA probe on the array, a signal derived from the label is obtained at a specific position on the array. Therefore, if the sequence and position of the DNA probe immobilized on the substrate is known in advance, nucleotide sequences existing in the sample gene can be examined in a simple manner. Also, if the DNA array is used, only one test makes it possible to obtain much information concerning nucleotide sequences. This is the reason why the DNA array is not limited to the gene screening technologies but is expected as sequencing technologies (Pease et al. 1994, Parinov et al. 1996).
On the other hand, with regard to the immobilization of a DNA probe to the substrate, two methods have been reported: (1) a method in which a DNA strand of a DNA probe is extended every single nucleotide one by one on the substrate (U.S. Pat. No. 5,889,165) and (2) a method in which a DNA probe synthesized in advance is immobilized to the substrate (U.S. Pat. No. 5,807,522). In the former method, DNA probes differing in sequence can be immobilized every 20xc3x9720 xcexcm at intervals of 100 xc3x85 on the substrate surface which is xc2xd by xc2xd inches square by utilizing the photolithographic technique, and therefore probe density of about 4xc3x97105 types of probe can be achieved (Chee et al. 1996). Because a patterning of about 0.1 xcexcm width is becoming possible at present by the photolithographic techniques, there is the possibility that probes are more integrated in the future. On the other hand, although the latter method has the problem that it is necessary to prepare many types of probe in advance and the degree of integration of probes is lower than that of the former method (every 60xc3x9760 xcexcm at intervals of 120 xcexcm), the latter method is superior in reaction efficiency because the probe can be immobilized three-dimensionally in a gel matrix (Guschin et al. 1997). Also, the latter method in which probes are immobilized three-dimensionally in a porous silicon rather than the gel has been reported (Beattie et al. 1995).
As outlined above, the DNA array has, for instance, an advantage that plural data are obtained in one assay. However, because complicated reaction control is required in the production of the DNA array, the conventional DNA array was highly expensive.
The present invention has been made to solve the aforementioned problems, and it is an object of the present invention to improve the conventional techniques used to produce a DNA array, thereby enabling to provide a nucleic acid immobilized carrier which has high cost performance, simplicity and sensitivity.
In the present invention, in order to attain the above objects, by using a first nucleic acid strand immobilized on a first substrate as so-called template, a second nucleic acid strand which is used as a probe nucleic acid strand is synthesized, and then, the resulting second nucleic acid strand is transferred to and immobilized on a second substrate by making use of an electric field, thereby producing a nucleic acid immobilized carrier.
Accordingly, the present invention provides a method for producing a nucleic acid immobilized carrier provided with a second nucleic acid strand of a predetermined sequence immobilized on, the method comprising the steps of:
preparing a first nucleic acid immobilized carrier which has a first nucleic acid strand immobilized on a first substrate, the first nucleic acid strand having a nucleotide sequence complementary to that of the second nucleic acid strand;
synthesizing a second nucleic acid strand along the first nucleic acid strand in a nucleic acid synthesizing solution, the second nucleic acid strand having a nucleotide sequence complementary to that of the first nucleic acid;
disposing a second substrate such that the second substrate faces toward one side of the first substrate, on which sides the first nucleic acid strand are immobilized; and
applying an electric field directed toward the first substrate from the second substrate to cause migration of the second nucleic acid strand, which has been synthesized along the first nucleic acid strand, onto the surface of the second substrate, thereby effecting immobilization of the second nucleic acid strand on the surface of the second substrate.
In the present invention, a pair of electrodes is used as means of applying the electric field. The electrodes are used by respectively disposing them outside of the first and the second substrates. Alternatively, instead of using the electrode separated from the substrate, the electrode and the substrate may be integrated into a composite electrode, which can be made by coating the first substrate and/or the second substrate with a conductive film.
In the present invention, a plurality of the first and the second nucleic acid strands may be used. In this case, although the nucleic acid strands may have the same sequences, it is preferable that these strands have a different sequence, respectively. In this case, when the composite electrode is used, it is preferred that the surface of the conductive film is divided into plural electrode regions by using insulation patterns, and a different nucleic acid probe strand is immobilized in each electrode region. Alternatively, different nucleic acid strand may be immobilized on the same electrode.
Additional objects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and obtained by means of the instrumentalities and combinations particularly pointed out hereinafter.